Fluorescent chromophores play an essential role in molecular imaging both in vitro and in vivo (Goncalves, M. S. Chem. Rev. 2009, 109, 190). Of these fluorescent reporters, far-red and near infrared (NIR) dyes have ideal absorption/emission wavelengths between 600 and 1000 nm, which minimize autofluorescence interference from tissue and have minimal overlap with biological chromophores such as haemoglobin (Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123). NIR fluorophores have become the best candidates for use in fluorescent tags and fluorogenic probes for in vivo imaging (Ballou, B.; Ernst, L. A.; Waggoner, A. S. Curr. Med. Chem. 2005, 12, 795; Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov, A., Jr. Nat. Biotechnol. 1999, 17, 375). For example, NIR dyes conjugated to peptides or nanoparticles have successfully been applied to in vivo imaging of tumors, (Ballou, B.; Ernst, L. A.; Waggoner, A. S. Curr. Med. Chem. 2005, 12, 795; Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov, A., Jr. Nat. Biotechnol. 1999, 17, 375; Weissleder. Nat. Biotechnol. 2001, 10, 316; Leimgruber, A. B. C.; Cortez-Retamozo, V.; Etzrodt, M.; Newton, A. P.; Waterman P.; Figueiredo, J. L.; Kohler, R. H.; Elpek, N.; Mempel, T. R.; Swirski, F. K.; Nahrendorf, M.; Weissleder, R.; Pittet, M. J. Neoplasia 2009, 11) myocardial infraction (Sosnovik, D.; Nahrendorf, M.; Weissleder, R. Nat. Clin. Pract. Card. 2008, 5, S63) and inflammation (Cortez-Retamozo, V.; Swirski, F. K.; Waterman, P.; Yuan, H.; Figueiredo, J. L.; Newton, A. P.; Upadhyay, R.; Vinegoni, C.; Kohler, R.; Blois, J.; Smith, A.; Nahredorf, M.; Josephson, L.; Weissleder, R.; Pittet, M. J. Clin. Invest. 2008, 118, 4058).
Carbocyanine fluorophores are one of the canonical NIR dye families with excellent optical properties, including tunable NIR emission, high extinction coefficients, and good fluorescence quantum yields (Hilderbrand, S. A.; Kelly, K. A.; Weissleder, R.; Tung, C. H. Bioconjug. Chem. 2005, 16, 1275). Since the 1980s, a variety of carboxylic acid derivatized carbocyanines have been prepared to meet the increasing demand for their use in bioconjugation and imaging applications (Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S. Bioconjug. Chem. 1996, 7, 356; Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S. Bioconjug. Chem. 1993, 4, 105). However, their widespread use is hindered by the high cost and limited availability of large quantities for many of these fluorescent labels. Most monofunctional carbocyanine dyes are asymmetric with the conjugation handle attached to one of the quaternary nitrogens of the indolium or benzo[e]indolium moieties. During the synthesis of these asymmetric dyes, undesired symmetric dyes are also formed (FIG. 1). These symmetric dye byproducts are often difficult to separate from the desired monofunctional dyes and contribute to decreased synthetic yields, often lower than 10% (Lin, Y.; Weissleder, R.; Tung, C. H. Bioconjug. Chem. 2002, 13, 605).